A high-throughput UPLC-MS-MS Bio-analytical method for the analysis of veterinary pharmaceutical residues in Chicken Tissues, Application of efficient-valid-green (EVG) Framework as a Competence Tool.

Antibacterial medications are receiving the most attention due to hypersensitivity reactions and the emergence of bacterial mutants resistant to antibiotics. Treating Animals with uncontrolled amounts of antibiotics will extend beyond their lives and affect humans. This study aims to determine the concentration of the residues of sulfadimidine, sulfaquinoxaline, diaveridine, and vitamin K3 in the tissues of poultry (muscles and liver) after treatment with the combined veterinary formulation. A UPLC-MS-MS method was developed using Poroshell 120 ECC18 and a mobile phase composed of acetonitrile and distilled water, containing 0.1 % formic acid, in the ratio of (85:15 v/v) at a flow rate of 0.6 mL/min. Sample extraction solvent was optimized using response surface methodology (RSM) to be acetonitrile: methanol in the ratio (49.8: 50.2 v/v), and the method was validated according to the FDA bioanalytical method validation protocol over the range (50-1000 µg/Kg) for sulfaquinoxaline and (50-750 µg/Kg) for the other 3 drugs. The greenness of the sample preparation and analytical method was assessed by applying Analytical Eco-scale (AES) and AGREE coupled with AGREEprep. The Competence of the study was evaluated via the EVG framework known as Efficiency, validation, and greenness, to achieve a balance point represented by a radar chart. The method was applied to decide the time required for poultry products to be safe for human use after administration of the studied drugs. It was found that, after the administration of the last dose, minimally 7 days are required till the levels of the drugs drop to the maximum residue limit determined by the FDA/WHO in animal tissues.

An integrated framework to develop an efficient valid green (EVG) HPLC method for the assessment of antimicrobial pollutants with potential threats to human health in aquatic systems.

The persistence of antimicrobial drugs in aquatic environments has raised critical concerns about their possible impact on drinkable water quality and human health. The Nile River is experiencing water pollution owing to increasing discharges of highly contaminated home and industrial effluents and inadequate water management systems. Investigations of the presence of three antimicrobial agents, ciprofloxacin (CIP), sulfamethoxazole (SMZ), and albendazole (ALB), in the Egyptian aquatic system are recommended using a chromatographic method because of their reported existence in the African aquatic environment. In this study, an integrated framework, Efficient Valid Green (EVG), for analytical techniques is proposed and displayed via its radar chart. The EVG framework is achieved through three main pillars: efficiency, validation, and greenness. The proposed EVG-HPLC method was developed and optimized using the AQbD methodology via a face-centered composite (FCC) design by identifying the proper critical method parameters (CMPs) that influence critical quality attributes (CQAs). The method was fully validated according to ICH guidelines, including a factorial robustness study within concentration ranges of 1-100 µg mL-1, 2-100 µg mL-1, and 10-100 µg mL-1 for CIP, SMZ, and ALB, respectively. The proposed method was evaluated in terms of greenness using AGREE (score 0.55) and ComplexGAPI metrics. The optimized chromatographic conditions included a C18 column and a mobile phase of water : acetonitrile : methanol in a ratio of 60 : 19 : 21, v/v/v, respectively, with an aqueous solution of pH 3.5 adjusted with phosphoric acid at a flow rate of 1.57 mL min-1 at 285 nm. The raw water samples collected from Nile River freshwater at different locations were treated using Oasis® PRiME HLB cartridges with satisfactory recoveries for the three analytes (>90%), and the three drugs were detected using the proposed EVG-HPLC method.

Green wastewater treatment of repurposed COVID-19 therapy (levofloxacin) using synthesized magnetite pectin nanoparticles, comparison with mesoporous silica nanoparticles.

RATIONALE: Antibiotics have been detected worldwide in the aquatic environment. Moreover, certain classes of antibiotics have been repurposed for the management of COVID-19, which increased their use and presence in wastewater. Their occurrence even in low concentrations leads to the development of antibiotic resistance. METHODOLOGY: Magnetite pectin nanoparticles (MPNP) were fabricated and compared to an established model of mesoporous silica nanoparticles (MSNP). Our studied adsorbate is levofloxacin, a fluoroquinolone antibiotic, commonly used in managing COVID-19 cases. RESULTS: The influence of various factors affecting the adsorption process was studied, such as pH, the type and concentration of the adsorbent, contact time, and drug concentration. The results illustrated that the optimum adsorption capacity for antibiotic clearance from wastewater using MPNP was at pH 4 with a contact time of 4 h; while using MSNP, it was found to be optimum at pH 7 with a contact time of 12 h at concentrations of 10 µg/mL and 16 g/L of the drug and nanoparticles, respectively, showing adsorption percentages of 96.55% and 98.89%. Drug adsorption equilibrium data obeyed the Sips isotherm model. DISCUSSION AND CONCLUSION: HPLC assay method was developed and validated. The experimental results revealed that the MPNP was as efficient as MSNP for removing the antibacterial agent. Moreover, MPNP is eco-friendly (a natural by-product of citrus fruit) and more economic as it could be recovered and reused. The procedure was evaluated according to the greenness assessment tools: AGREE calculator and Hexagon-CALIFICAMET, showing good green scores, ensuring the process's eco-friendliness.

Response surface optimization of a cardioprotective compound through pharmacosomal drug delivery system: in vivo bioavailability and cardioprotective activity potential.

Vanillic acid (VA) is a phenolic compound with potential antioxidant activity, which improves ischemia-induced myocardial degeneration, by reducing oxidative stress; however, it suffers poor bioavailability owing to its poor solubility. VA-loaded pharmacosomes were optimized using a central composite design, where the effect of phosphatidylcholine:VA molar ratio and the precursor concentration were studied. An optimized formulation (O1) was prepared and tested for the release rate of VA, in vivo bioavailability, and cardioprotective potential on myocardial infarction-induced rats. The optimized formulation showed a particle size of 229.7 nm, polydispersity index of 0.29, and zeta potential of - 30 mV. O1 showed a sustained drug release for 48 h. The HPLC-UV method was developed for the determination of VA in plasma samples using protein precipitation. The optimized formulation showed a great improvement in the bioavailability as compared to VA. The residence time of the optimized formula was 3 times longer than VA. The optimized formulation showed a more potent cardioprotective effect as compared to VA, via inhibition of the MAPK pathway with subsequent inhibition of PI3k/NF-κB signaling, in addition to its antioxidant effect. The optimized formulation showed normalization of many oxidative stress and inflammatory biomarkers. Thus, a VA-loaded pharmacosome formulation with promising bioavailability and cardioprotective activity potential was prepared.

The power of High Impact Amplitude Manipulation (HIAM) technique for extracting the basic spectra of two Fixed-dose combinations (FDC) -Spectrophotometric purity analysis via spectral contrast angle.

HIAM technique allows the extraction of the original constant signal of each single component out of interference signals of a mixture and further transformed into basic spectrum (D0). It includes the methods: ratio subtraction coupled with unified constant subtraction (RS-UCS), constant center (CC) and constant extraction (CE). The technique was introduced for the analysis of two pharmaceutical formulations used to treat cardiovascular diseases. The formulations are binary combinations of Amlodipine (AML) with either Atorvastatin (ATR) or Candesartan (CND) which shows interefernce absorbance signals. The technique was valid over the linearity range of (5.0-35.0 µg/ml) for AML, ATR and CND with recovery percentage 100.40 ± 1.88 , 100.00 ± 0.86 and 99.83 ± 1.07, respectively . The extracted signals were tested for its purity by spectral contrast angle (cos θ) to illustrate the efficency of the HIAM technique where cos θ values ranges from (0.9902 to 0.9986). The presented technique was fully validated regarding ICH guidelines and were statistically compared using one-way ANOVA at 95% confidence.

Appraisal for Edible Use of Vegetable Crops Cultivated in Egypt after Treatment with Selected Insecticides and Fungicides: Insights of Dissipation Rates and Pre-harvest Intervals.

An analytical investigation was carried out to study the dissipation rate of two commonly used pesticides, thiamethoxam (neonicotinoid insecticide) and propamocarb hydrochloride (carbamate fungicide), applied to four vegetable crops: cucumber, zucchini, lettuce and pepper, after open-field application. Samples were harvested according to a scheduled plan followed by QuEChERS extraction, then thiamethoxam residues were analyzed using a GC-ECD method, while propamocarb HCl residues were analyzed using an HPLC-UV method. Validation parameters were attained for both methods and the kinetic profile was studied, which fitted the first-order kinetics where k, t1/2 and t90 were calculated. The proper pre-harvest interval (PHI) was studied for each crop to ensure that the residues levels declined to reach below the maximum residue limit (MRL) where the crop is suitable for consumption. These values were found to be different from labelled values, which proves that the PHIs are greatly affected by changing weather conditions.

Eco-friendly UPLC-MS/MS analysis of possible add-on therapy for COVID-19 in human plasma: Insights of greenness assessment.

Facing the pandemic COVID-19 is of highest priority for all researchers nowadays. Recent statistics indicate that the majority of the cases are home-treated. Two drugs of interest, Guaifenesin and Bromohexine HCl, are among the add-on therapy for treatment of COVID-19 mild cases, which has raised the need for their simultaneous determination. The analysis of the two drugs of interest was described using ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) in plasma of healthy human volunteers using tetryzoline HCl as an internal standard (IS) after liquid-liquid extraction. The applied chromatographic conditions were Kinetex C18 (100 Å, 2.6 µm X 50 mm X 4.6 mm) column and a mixture of methanol: water (95: 5, v/v) as a mobile phase at flow rate 1 mL/min. The positive ionization mode was used for detecting the ions, by observing the pairs of transition m/z 199 < 125 for GUF, m/z 377 < 114 for BRM and m/z 201 < 131 for IS. The linearity range was from 50 to 1500 ng/mL for GUF and 0.5-50 µg/mL for BRM. Limit of detection (LOD) was found to be 35.16 and 0.43 ng/ml for GUF and BRM, respectively. The method was validated according to FDA guidance. The proposed method was assessed to be more eco-friendly versus the reported method using the greenness assessment tools: National Environmental Methods Index (NEMI), Assessment of Green Profile (AGP), Green Analytical Procedure Index (GAPI) and Eco-Scale. The proposed method was applied for the application of a pilot pharmacokinetic study.

Trade-off efficacy and data processing strategy in the power of spectral resolution of co-formulated antihypertensive pharmaceuticals.

Versatile, extraction-free univariate spectrophotometric methods have been modified to get effective spectral resolution of mixtures in accordance with their feature challenges. The proposed methods have been applied and validated for analyzing some antihypertensive medicines in their co-formulated medicinal products. Two mixtures have been used: the first one [Mix I (OLM/ADB)] is composed of Olmesartan medoxomil (OLM) and Amlodipine besylate (ADB) with partly-overlapped spectra while the second [Mix II (TEL/HCT)] is made up Telmisartan (TEL) and Hydrochlorothiazide (HCT) with total-overlapped spectra. Induced dual wavelength, absorbance correction and ratio subtraction coupled with constant multiplication methods were applied to Mix I (OLM/ADB), while dual wavelength, advanced absorbance subtraction and constant center coupled with spectrum subtraction methods were applied to Mix II (TEL/HCT). Calibration graphs were established with good correlation coefficients. The methods exhibit significant advantages as simplicity, sensitivity, minimal data manipulation besides optimum robustness. Selectivity was inspected using lab-mixtures with diverse ratios. Accuracy, precision and repeatability were found to be within the acceptable limits. The proposed methods are good enough to be used for co-assay of analytes in combined forms without any interfering from excipients. Moreover, all results were estimated in accordance with ICH criteria and successfully compared with those of the reported methods applying t-test and F-test at 95% confidence level. Generally, these methods can be used efficiently for routine quality control testing.

Advanced approaches for the treatment and amplification of weak spectral signals produced by critical concentrations in white multicomponent systems.

An analytical investigation was carried out to study the treatment and amplification of the spectral signals produced by critical concentrations with high accuracy and precision using two advanced approaches. The factorized-spectrum approach was applied through two novel methods which were: absorptivity centering technique via both: factorized zero order absorption spectrum (ACT-FSD0ΔA) and factorized ratio spectrum (ACT-FSRΔP). The proposed methods were found to be linear in the ranges of (15-100 µg/mL) and (3-40 µg/mL) for ASP and MTO, respectively. Those methods were compared to the methods following the geometrical standard addition approach: ratio H-point standard addition method (RHPSAM) and geometrical induced amplitude modulation (GIAM). The approaches were applied for the determination of the minor component metoclopramide in its mixture with the major component aspirin in the challengeable ratio of (1,90) respectively in a white multicomponent system. The results obtained from the proposed approaches were statistically compared with each other. The methods were validated according to ICH guidelines where the results were found to be within the acceptable limits. The methods were found to be accurate and reliable for the determination of metoclopramide critical concentration besides aspirin concentration. The results of single factor ANOVA analysis indicated that there is no significant difference among the developed methods. These methods provided simple resolution of this binary combination from synthetic mixtures and pharmaceutical preparation and can be conveniently adopted for routine quality control analysis.

Investigating Advanced Approaches Based on Iso-Absorptivity Coefficient in Unresolved Spectral Signals of Binary Mixtures.

Several spectrophotometric approaches utilize different functions of the iso-absorptivity coefficient in zero-order absorption signals and its manipulated spectra. This work introduced an investigation concerning the efficiency power of recent methods based on iso-absorptivity coefficient in different spectral signals. These methods were as follows: absorptivity centering method (a-Centering), absorbance subtraction method (AS), amplitude modulation method (AM,) and amplitude summation method (A-Sum). These methods were applied to determine the binary mixture of ofloxacin (OFX) and dexamethasone (DXM). Linearity of the proposed methods was investigated in the range of 1.0-10.0 µg/ml for both drugs. The proposed methods were validated as per ICH guidelines and were successfully applied for the simultaneous determination of OFX and DXM in their pharmaceutical preparation without interference from additives. Statistical analysis of the results obtained by the proposed spectrophotometric methods compared with a reported method revealed no significant difference between the proposed and reported methods, confirming accuracy and precision at 95% confidence limit.

Testing the purity of spectral profiles: Finger-print resolution of complex matrices and extraction of absorbance signals.

The application of spectrophotometric techniques has shown a tremendous development over the past few years, where it is possible to determine the concentrations of several components in complex matrix. A new feature will be introduced in this work where the application of spectrophotometric techniques will be enhanced to resolution and checking the purity of signals. The finger-print resolution "ratio subtraction method" (RSM) was coupled with the novel complementary method "unified constant subtraction" (UCS); in addition to the methods: extended ratio subtraction method (EXRSM), constant multiplication (CM) or "spectrum subtraction" (SS). These techniques were applied for the determination of the complex matrix of the binary mixture of chloramphenicol and dexamethasone. By applying official spectrophotometric methods, direct determination of the components was allowed with no need for validation procedures. The spectrophotometric techniques were successfully applied to the laboratory prepared mixtures and the combined dosage form where the purity of the extracted signals were tested by calculating the spectral contrast angle (θ) and the spectral ratio factor (SRF) where the results were compared to show the capability to recover pure spectral profiles and detect the presence of impurities. The proposed methods proved that spectrophotometric techniques can be used for identification and separation of signals, similar to chromatographic techniques.

Development and validation of impurity-profiling UPLC method for the determination of sodium cromoglicate and tetryzoline hydrochloride: Application on rabbit aqueous humor.

Sodium cromoglicate (SCG), antihistaminic agent, and tetryzoline hydrochloride (TZH), a sympathomimetic agent, are formulated together as an ophthalmic preparation. An ultra-performance liquid chromatographic method with UV detection (UPLC-UV) was developed and validated for the quantitative determination of SCG and TZH in rabbit aqueous humor. Due to the instability of both SCG and TZH under alkaline conditions, the UPLC method was applied for their determination in the presence of their possible degradation impurities. The separation was performed using C18 column (1.7µm particle size) and isocratic elution system with methanol: 1% o-phosphoric acid (65: 35, v/v).The optimum flow rate was 0.5ml/min and the detection was done at 230nm. The suggested method was validated in compliance with the ICH guidelines and was successfully applied for determination of sodium cromoglicate (SCG) and tetryzoline HCl (TZH) as prepared synthetically in laboratory mixtures, and in the presence of their alkali-induced degradation impurities. The suggested method was effectively applied the determination of spiked rabbit aqueous humor samples as well as commercial pharmaceutical formulation.

Computation of geometric representation of novel spectrophotometric methods used for the analysis of minor components in pharmaceutical preparations.

Novel spectrophotometric methods were applied for the determination of the minor component tetryzoline HCl (TZH) in its ternary mixture with ofloxacin (OFX) and prednisolone acetate (PA) in the ratio of (1:5:7.5), and in its binary mixture with sodium cromoglicate (SCG) in the ratio of (1:80). The novel spectrophotometric methods determined the minor component (TZH) successfully in the two selected mixtures by computing the geometrical relationship of either standard addition or subtraction. The novel spectrophotometric methods are: geometrical amplitude modulation (GAM), geometrical induced amplitude modulation (GIAM), ratio H-point standard addition method (RHPSAM) and compensated area under the curve (CAUC). The proposed methods were successfully applied for the determination of the minor component TZH below its concentration range. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed. No difference was observed between the obtained results when compared to the reported HPLC method, which proved that the developed methods could be alternative to HPLC techniques in quality control laboratories.

Novel two wavelength spectrophotometric methods for simultaneous determination of binary mixtures with severely overlapping spectra.

This work presents the application of different spectrophotometric techniques based on two wavelengths for the determination of severely overlapped spectral components in a binary mixture without prior separation. Four novel spectrophotometric methods were developed namely: induced dual wavelength method (IDW), dual wavelength resolution technique (DWRT), advanced amplitude modulation method (AAM) and induced amplitude modulation method (IAM). The results of the novel methods were compared to that of three well-established methods which were: dual wavelength method (DW), Vierordt's method (VD) and bivariate method (BV). The developed methods were applied for the analysis of the binary mixture of hydrocortisone acetate (HCA) and fusidic acid (FSA) formulated as topical cream accompanied by the determination of methyl paraben and propyl paraben present as preservatives. The specificity of the novel methods was investigated by analyzing laboratory prepared mixtures and the combined dosage form. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed. No difference was observed between the obtained results when compared to the reported HPLC method, which proved that the developed methods could be alternative to HPLC techniques in quality control laboratories.

Column performance study of different variants of liquid chromatographic technique: an application on pharmaceutical ternary mixtures containing tetryzoline.

High-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and rapid resolution liquid chromatographic (RRLC) methods have been developed and validated for the separation and quantitation of both or either of two ternary mixtures present in ophthalmic solutions. The first mixture contains chloramphenicol, dexamethasone sodium phosphate and tetryzoline HCl (TZH); while the second one contains ofloxacin, prednisolone acetate and TZH. Both preparations contain benzalkonium chloride as a preservative. The columns used were a HPLC column (C18 5 µm particle size), a RRLC column (C18 2.6 µm particle size) and a UPLC column (C18 1.7 µm particle size). A comparative study was conducted to illustrate the effect of the change in column particle size and dimensions on the other chromatographic conditions, backpressure and the separation of both ternary mixtures. The methods were validated as per ICH guidelines where accuracy, repeatability, interday precision and robustness were found to be within the acceptable limits. The RRLC column provided shorter run time and better resolution than HPLC, while the UPLC column gave the shortest run time for all columns. The RRLC column resulted in minimum backpressure, so it could be used with any HPLC instrument, which makes the method more practical and economic. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed.

A comparative study of different aspects of manipulating ratio spectra applied for ternary mixtures: derivative spectrophotometry versus wavelet transform.

This work represents a comparative study of different aspects of manipulating ratio spectra, which are: double divisor ratio spectra derivative (DR-DD), area under curve of derivative ratio (DR-AUC) and its novel approach, namely area under the curve correction method (AUCCM) applied for overlapped spectra; successive derivative of ratio spectra (SDR) and continuous wavelet transform (CWT) methods. The proposed methods represent different aspects of manipulating ratio spectra of the ternary mixture of Ofloxacin (OFX), Prednisolone acetate (PA) and Tetryzoline HCl (TZH) combined in eye drops in the presence of benzalkonium chloride as a preservative. The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulation containing the cited drugs. The proposed methods were validated according to the ICH guidelines. A comparative study was conducted between those methods regarding simplicity, limitation and sensitivity. The obtained results were statistically compared with those obtained from the reported HPLC method, showing no significant difference with respect to accuracy and precision.

A comparative study of progressive versus successive spectrophotometric resolution techniques applied for pharmaceutical ternary mixtures.

This work represents a comparative study of a novel progressive spectrophotometric resolution technique namely, amplitude center method (ACM), versus the well-established successive spectrophotometric resolution techniques namely; successive derivative subtraction (SDS); successive derivative of ratio spectra (SDR) and mean centering of ratio spectra (MCR). All the proposed spectrophotometric techniques consist of several consecutive steps utilizing ratio and/or derivative spectra. The novel amplitude center method (ACM) can be used for the determination of ternary mixtures using single divisor where the concentrations of the components are determined through progressive manipulation performed on the same ratio spectrum. Those methods were applied for the analysis of the ternary mixture of chloramphenicol (CHL), dexamethasone sodium phosphate (DXM) and tetryzoline hydrochloride (TZH) in eye drops in the presence of benzalkonium chloride as a preservative. The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulation containing the cited drugs. The proposed methods were validated according to the ICH guidelines. A comparative study was conducted between those methods regarding simplicity, limitation and sensitivity. The obtained results were statistically compared with those obtained from the official BP methods, showing no significant difference with respect to accuracy and precision.

A comparative study of novel spectrophotometric methods based on isosbestic points; application on a pharmaceutical ternary mixture.

This work represents the application of the isosbestic points present in different absorption spectra. Three novel spectrophotometric methods were developed, the first method is the absorption subtraction method (AS) utilizing the isosbestic point in zero-order absorption spectra; the second method is the amplitude modulation method (AM) utilizing the isosbestic point in ratio spectra; and third method is the amplitude summation method (A-Sum) utilizing the isosbestic point in derivative spectra. The three methods were applied for the analysis of the ternary mixture of chloramphenicol (CHL), dexamethasone sodium phosphate (DXM) and tetryzoline hydrochloride (TZH) in eye drops in the presence of benzalkonium chloride as a preservative. The components at the isosbestic point were determined using the corresponding unified regression equation at this point with no need for a complementary method. The obtained results were statistically compared to each other and to that of the developed PLS model. The specificity of the developed methods was investigated by analyzing laboratory prepared mixtures and the combined dosage form. The methods were validated as per ICH guidelines where accuracy, repeatability, inter-day precision and robustness were found to be within the acceptable limits. The results obtained from the proposed methods were statistically compared with official ones where no significant difference was observed.

A comparative study of validated spectrophotometric and TLC- spectrodensitometric methods for the determination of sodium cromoglicate and fluorometholone in ophthalmic solution.

The determination of sodium cromoglicate (SCG) and fluorometholone (FLU) in ophthalmic solution was developed by simple, sensitive and precise methods. Three spectrophotometric methods were applied: absorptivity factor (a-Factor method), absorption factor (AFM) and mean centering of ratio spectra (MCR). The linearity ranges of SCG were found to be (2.5-35 µg/mL) for (a-Factor method) and (MCR); while for (AFM), it was found to be (7.5-50 µg/mL). The linearity ranges of FLU were found to be (4-16 µg/mL) for (a-Factor method) and (AFM); while for (MCR), it was found to be (2-16 µg/mL). The mean percentage recoveries/RSD for SCG were found to be 100.31/0.90, 100.23/0.57 and 100.43/1.21; while for FLU, they were found to be 100.11/0.56, 99.97/0.35 and 99.94/0.88 using (a-Factor method), (AFM) and (MCR), respectively. A TLC-spectrodensitometric method was developed by separation of SCG and FLU on silica gel 60 F254 using chloroform:methanol:toluene:triethylamine in the ratio of (5:2:4:1 v/v/v/v) as developing system, followed by spectrodensitometric measurement of the bands at 241 nm. The linearity ranges and the mean percentage recoveries/RSD were found to be (0.4-4.4 µg/band), 100.24/1.44 and (0.2-1.6 µg/band), 99.95/1.50 for SCG and FLU, respectively. A comparative study was conducted between the proposed methods to discuss the advantage of each method. The suggested methods were validated in compliance with the ICH guidelines and were successfully applied for the determination of SCG and FLU in their laboratory prepared mixtures and commercial ophthalmic solution in the presence of benzalkonium chloride as a preservative. These methods could be an alternative to different HPLC techniques in quality control laboratories lacking the required facilities for those expensive techniques.